Loss of selenium from selenoproteins: conversion of selenocysteine to dehydroalanine in vitro

Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-beta-elimination of selenenic acid during sample processing.     

Post-transcriptional control of selenoprotein biosynthesis

Selenoproteins are defined as proteins containing the 21st proteinogenic amino acid, selenocysteine (Sec). Sec is encoded by UGA (STOP) codons which are re-coded to Sec by the presence of a selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region of selenoprotein mRNAs. The SECIS element is bound by several proteins, including SECIS-binding protein 2 (SBP2). Translation of selenoproteins critically depends on the integrity of the SECIS element - SBP2 interaction. Mutations in a SECIS element can abrogate expression of the respective selenoprotein. Mutations in SBP2 impinge on biosynthesis of a subset of selenoproteins and lead to a syndrome including hormonal, neurological, immunological symptoms as well as myopathy. Several other RNA-binding proteins are involved in selenoprotein translation and mediate the hierarchical response of selenoproteins to selenium deficiency. Global inhibition of selenoprotein translation is lethal in the mouse and hypomorphic mutations in selenocysteine synthase in humans leads to Progressive Cerebello Cerebral Atrophy, a neurodevelopmental and neurodegenerative disease in pediatric patients.     

Fractionation of selenium-containing proteins in serum by multiaffinity liquid chromatography before size-exclusion chromatography–ICPMS

Immunoaffinity chromatography has been investigated for fractionation of serum into selenoalbumin and true selenoproteins. Among several albumin-depletion kits tested, a multiaffinity column specifically binding albumin and five other major serum proteins provided the best results. It extracted ca 95% of both albumin and selenoalbumin, which enabled interference-free determination of glutathione peroxidase, selenoprotein P, and selenoalbumin by size-exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC–ICPMS). The efficiency of the multiaffinity column did not vary over a period of 18 months. The purity of fractions separated by immunoaffinity LC was confirmed by elution-volume matching with standards in SEC–ICPMS and by selenopeptide mapping in capillary HPLC–ICPMS. Quantification of the selenium distribution among the different proteins in human serum from a control group and from a person on a selenium-rich diet revealed that 67% of the supplemented selenium was incorporated into albumin, 30% into glutathione peroxidase, and 3% into selenoprotein P.     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency

Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides, and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes, and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. To fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved (11)CXX(14)C active site were chemically synthesized. Grx3, Grx3(C11U), and Grx3(C14U) exhibited redox potentials of -194, -260, and -275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (-309 mV) and thioredoxin (Trx) (-270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a "central atom" in the exchange reaction. The 10(2)-10(4)-fold increase in the rate of Trx reduction by the seleno-Grx3 analogues demonstrates that oxidoreductases containing either selenenyl-sulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts.     

Selenoproteins: the key factor in selenium essentiality. State of the art analytical techniques for selenoprotein studies

Selenium is an essential element for human health. The benefits of selenium are many including protection against cancer, heart diseases and other cardiovascular and muscle disorders. Selenium is also helpful in controlling gastrointestinal disorders, enhancing immunity of the human body and reducing age-related diseases. The health-promoting properties of Se are due to vital functions of selenoproteins in which selenium is present as selenocysteine, the 21st amino acid. To date, dozens of selenoprotein families have been described though many have roles that have not been fully elucidated. Selenoproteins research has attracted tremendous interest from different scientific areas. Analytical chemists have not remained indifferent to the attractive features of these unique proteins. Different analytical techniques, such as multidimensional chromatography–inductively coupled plasma mass spectrometry (ICPMS), electrospray (tandem) mass spectrometry (ESI-MS/MS), matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis–laser ablation inductively coupled plasma mass spectrometry (SDS-PAGE-LA-ICPMS), have been applied to the determination of selenoproteins and selenium-containing proteins. This review describes the best-characterized selenoproteins to date in addition to the major contributions of analytical chemistry to the field of selenoproteins. The article also highlights the challenges of combining elemental and molecular mass spectrometry for the determination of selenoproteins and selenium-containing proteins.     

Protein factors mediating selenoprotein synthesis

The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF-Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis.     

Reversed-phase liquid chromatography in-line with negative ionization electrospray mass spectrometry for the characterization of the disulfide-linkages of an immunoglobulin gamma antibody

In this report, we present a new approach for the determination of the disulfide bond connectivity in proteins using negative ionization mass spectrometry of nonreduced enzymatic digests. The mass spectrometric analysis in negative ion mode was optimized to allow in-line analysis coupled directly to the HPLC system used for the separation of the peptides resulting from enzymatic digestion. We determined the disulfide structure of a human immunoglobulin gamma 2 (IgG2) antibody containing 18 unique cysteine residues linked via 11 unique disulfide bonds. The efficiency of the gas-phase dissociation of disulfide-linked peptides using negative electrospray ionization was evaluated for an ion trap mass spectrometer and an orthogonal acceleration time-of-flight mass spectrometer. Both mass spectrometry techniques provided efficient in-source fragmentation for the identification of the disulfide-linked peptides of the antibody. Both instruments were limited in the number of disulfide bonds that could be dissociated. Seven of the 11 unique disulfide linkages have been determined, including the linkage of the light chain to the heavy chain. Only the disulfide connectivity of the hinge peptide H6H7H8H9 (C6C7VEC8PPC9PAPPVAGPSVFLFPPKPK) could not be determined (numbering the cysteine residues sequentially from the N-terminus and labeling the heavy chain cysteines "H" and the light chain cysteines "L"). However, we identified the dimer of peptide C6C7VEC8PPC9PAPPVAGPSVFLFPPKPK linked via four disulfide bonds based on the unique molecular weight of this dipeptide. The established linkages were H1 to H2, H10 to H11, H12 to H13, L1 to L2, L3 to L4, and L5 to H3H4. The intrachain linkages of the light chain (L1 to L2, L3 to L4), and heavy chain (H10 to H11, H12 to H13) domains were identical to the linkages found in IgG1 antibodies.     

Mechanisms Affecting the Biosynthesis and Incorporation Rate of Selenocysteine

Selenocysteine (Sec) is the 21st non-standard proteinogenic amino acid. Due to the particularity of the codon encoding Sec, the selenoprotein synthesis needs to be completed by unique mechanisms in specific biological systems. In this paper, the underlying mechanisms for the biosynthesis and incorporation of Sec into selenoprotein were comprehensively reviewed on five aspects: (i) the specific biosynthesis mechanism of Sec and the role of its internal influencing factors (SelA, SelB, SelC, SelD, SPS2 and PSTK); (ii) the elements (SECIS, PSL, SPUR and RF) on mRNA and their functional mechanisms; (iii) the specificity (either translation termination or translation into Sec) of UGA; (iv) the structure–activity relationship and action mechanism of SelA, SelB, SelC and SelD; and (v) the operating mechanism of two key enzyme systems for inorganic selenium source flow before Sec synthesis. Lastly, the size of the translation initiation interval, other action modes of SECIS and effects of REPS (Repetitive Extragenic Palindromic Sequences) that affect the incorporation efficiency of Sec was also discussed to provide scientific basis for the large-scale industrial fermentation for the production of selenoprotein.     

Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein

The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-alpha 1-6-mannose-beta 1-4-N-acetylglucosamine-N-acetylglucosamine(alpha 1-6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. (c) 2000 John Wiley & Sons, Ltd.     

Structural analysis of saponins from medicinal herbs using electrospray ionization tandem mass spectrometry

The underivatized saponins from Tribulus terrestris and Panax ginseng have been investigated by electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). In ESI-MS spectra, a predominant [M + Na](+) ion in positive mode and [M - H](-) ion in negative mode were observed for molecular mass information. Multi-stage tandem mass spectrometry of the molecular ions was used for detailed structural analysis. Fragment ions from glycoside cleavage can provide information on the mass of aglycone and the primary sequence and branching of oligosaccharide chains in terms of classes of monosaccharides. Fragment ions from cross-ring cleavages of sugar residues can give some information about the linkages between sugar residues. It was found that different alkali metal-cationized adducts with saponins have different degrees of fragmentation, which may originate from the different affinity of a saponin with each alkali metal in the gas phase. ESI-MS(n) has been proven to be an effective tool for rapid determination of native saponins in extract mixtures, thus avoiding tedious derivatization and separation steps.     

Permethylation Linkage Analysis Techniques for Residual Carbohydrates

Permethylation analysis is the classic approach to establishing the position of glycosidic linkages between sugar residues. Typically, the carbohydrate is derivatized to form acid-stable methyl ethers, hydrolyzed, peracetylated, and analyzed by gas chromatography-mass spectrometry. The position of glycosidic linkages in the starting carbohydrate are apparent from the mass spectra as determined by the location of acetyl residues. The completeness of permethylation is dependent upon the choice of base catalyst and is readily confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry mass spectrometry. For the permethylation of β-cyclodextrin, Hakomori dimsyl base is shown to be superior to the NaOH–dimethyl sulfoxide system, and the use of the latter resulted in selective under-methylation of the 3-hydroxy groups. These techniques are highly applicable to residual carbohydrates from biofuel processes. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Diagnostic ions for the rapid analysis by nano-electrospray ionization quadrupole time-of-flight mass spectrometry of O-glycans from human mucins

Nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-Q-TOFMS) was used for sensitive mapping and sequencing of underivatized oligosaccharide alditols obtained from human mucins. Using subnanomolar amounts of oligosaccharides previously analyzed by nuclear magnetic resonance (NMR), series of diagnostic ions relevant to the structural characterization of O-glycans were deduced. Determination of the core type as well as positions and partial linkages of fucose residues could be readily obtained from the dominant [M+Na](+) ions. Differentiation of isomeric structures and glycosidic linkages were defined by the characteristic cross-ring (0,2)A-type cleavages in the negative ion mode. Tandem (MS/MS) mass spectra of [M-H](-) ions from sialylated or sulfated O-glycans revealed information concerning the position and linkage of such residues. These fragmentation rules were further applied in the structural determination of glycans from human colonic mucins. All these findings indicated the efficiency of ESI-Q-TOFMS for the determination of oligosaccharide composition, sequence, partial linkage and substitution, providing a wealth of structural information with sensitivity sufficient for the analysis of quantities as obtained from natural sources.     

Internally stabilized selenocysteine derivatives: syntheses, 77Se NMR and biomimetic studies

Selenocystine ([Sec]2) and aryl-substituted selenocysteine (Sec) derivatives are synthesized, starting from commercially available amino acid l-serine. These compounds are characterized by a number of analytical techniques such as NMR (1H, 13C and 77Se) and TOF mass spectroscopy. This study reveals that the introduction of amino/imino substituents capable of interacting with selenium may stabilize the Sec derivatives. This study further suggests that the oxidation-elimination reactions in Sec derivatives could be used for the generation of biologically active selenols having internally stabilizing substituents.     

Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods

Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.     

Detection of selenocompounds in a tryptic digest of yeast selenoprotein by MALDI time-of-flight MS prior to their structural analysis by electrospray ionization triple quadrupole MS

MALDI-TOFMS was proposed as a key technique to a novel generic approach for the speciation analysis of selenium in yeast supplements. Owing to a lower detection limit and superior matrix tolerance to electrospray MS it allowed a successful detection of selenocompounds in samples for which electrospray MS had failed. The analytical approach developed was applied to the identification of a previously unreported selenopentapeptide (m/z 596) in the tryptic digest of a water-soluble selenoprotein fraction isolated by size-exclusion chromatography. The information on the mass of the protonated molecular ion obtained from MALDI allowed the optimization of the conditions for collision induced dissociation MS using a triple quadrupole spectrometer that enabled the determination of the amino acid sequence SeMet-Asn-Ala-Gly-Arg of the selenopeptide.     

Differentiating structural isomers of sialylated glycans by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry

Using model acidic glycans, we demonstrate the benefits of permethylation for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) tandem mass spectrometry. With both the linear and branched structures, extensive cross-ring fragmentation product ions were generated, yielding valuable information on sugar linkages. Elimination of the negative charges commonly associated with sialylated structures through permethylation allowed their structural analysis in the positive ion mode. Extensive A- and X-type ions were observed for the linear structures, and slightly weaker signals for the branched sialylated structures. The diagnostic cross-ring fragments, permitting a distinction between alpha2-3 and alpha2-6 linkages of the sialic acid residues, were seen in abundance. Importantly, the cross-ring fragmentation with the branched structures provides adequate information to assign sialic acid residues, with a specific linkage, to a particular antenna.     

MALDI‐FT‐ICR‐MS for archaeological lipid residue analysis

Soft‐ionization methods are currently at the forefront of developing novel methods for analysing degraded archaeological organic residues. Here, we present little‐used soft ionization method of matrix assisted laser desorption/ionization‐Fourier transform‐ion cyclotron resonance‐mass spectrometry (MALDI‐FT‐ICR‐MS) for the identification of archaeological lipid residues. It is a high‐resolution and sensitive method with low limits of detection capable of identifying lipid compounds in small concentrations, thus providing a highly potential new technique for the analysis of degraded lipid components. A thorough methodology development for analysing cooked and degraded food remains from ceramic vessels was carried out, and the most efficient sample preparation protocol is described. The identified components, also controlled by independent parallel analysis by gas chromatography‐mass spectrometry (GC‐MS) and gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), demonstrate its capability of identifying very different food residues including dairy, adipose fats as well as lipids of aquatic origin. The results obtained from experimentally cooked and original archaeological samples prove the suitability of MALDI‐FT‐ICR‐MS for analysing archaeological organic residues. Sample preparation protocol and identification of compounds provide future reference for analysing various aged and degraded lipid residues in different organic and mineral matrices.     

High-performance liquid chromatography--mass spectrometry and electron-capture dissociation tandem mass spectrometry of osteocalcin. Determination of gamma-carboxyglutamic acid residues

Two mass spectrometry methods, high-performance liquid chromatography combined on-line with electrospray ionization mass spectrometry (HPLC-ESI-MS) and electron-capture (EC) dissociation tandem mass spectrometry (MS-MS), were applied for structural analysis of bovine and human osteocalcins. Osteocalcin contains gamma-carboxyglutamic acid (Gla) residues, which bind metal ions, among its amino acids. Ethylenediaminetetraacetic acid (EDTA) was added to all samples in order to chelate bound metal ions. After elimination of interfering metal ions MS spectra became uncomplicated to interpret. EDTA is incompatible with ESI and it was removed from samples using either on-line HPLC or micropurification method. The number of Gla residues varies in osteocalcin. These subforms, which contain different amounts of Gla residues, were separated using the HPLC-ESI-MS method. In order to determine locations of Gla residues in human osteocalcin, which contained two Gla residues, dissociation MS-MS method was successfully applied.     

Electrospray ionization ion trap mass spectrometry for structural characterization of oligosaccharides derivatized with 2-aminobenzamide

The use of electrospray ionization (ESI) quadrupole ion trap mass spectrometry and reversed-phase high-performance liquid chromatography (HPLC) for the characterization of 2-aminobenzamide (2AB)-labeled oligosaccharides and N-linked protein oligosaccharide mixtures is described. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2AB-derivatized oligosaccharides. Under collision-induced dissociation, sodiated molecular species generated in the ESI mode yield simple and predictable mass spectra. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Information on composition, sequence, branching and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds and from weak cross-ring cleavage products. Reversed-phase HPLC and derivatization by reductive amination using 2-aminobenzamide were finally applied to characterize a glycan pool enzymatically released from glycoproteins.